6. DISCUSSION
6.4. suPAR as a prognostic factor in Staphylococcus aureus bacteraemia
with a fatal outcome than survivors in 30-day surveillance period. Day three, four and 10 suPAR levels were higher in fatalities than in survivors, whereas suPAR levels in patients with a deep infection focus did not differ from those without a deep infection focus. In contrast to suPAR, CRP on day three was not prognostic for fatal outcome. suPAR and CRP levels were not evaluated on the positive blood culture day. However, in the entire 430 SAB material (Study II), CRP levels on the positive blood culture day and on day three were evaluated, and found non-prognostic for mortality. Consistent with our study, high suPAR levels appeared to be prognostic for fatal outcome in other bacteraemia studies on
Streptococcus (pneumonia and β-haemolytic) and E. coli [389,390]. In critically ill septic
patients, low suPAR levels are a positive predictor of overall survival [391]. In our study, we observed that suPAR levels on day 10 could predict mortality, which is consistent with findings of suPAR being prognostic in serial measurements in septic ICU patients up to 10- 14 days [470,471].
In our study, presence of a deep infection focus was not associated with elevated suPAR levels. Publications on suPAR and diagnosis of deep infection in SAB are not available. In
Streptococcus pneumonia bacteraemia study, patients with deep infection were not
included [389] and in a report on pneumonia and meningitis and use of suPAR, the impact of a deep infection focus on suPAR levels was not analysed [472]. Deep infection focus is very common in SAB (84% prevalence in our study), whereas mortality due to SAB is generally lower (about 20%) [159,207,284,458]. Our small pilot study suggests that suPAR might not be a suitable diagnostic marker for detecting a deep infection focus. As a
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diagnostic marker for infection, however, suPAR is useful when measured simultaneously with PCT. This combination could improve the efficiency of sepsis diagnosis, and the combination of plasma suPAR and APACHE II score could lead to more accurate mortality prediction [473].
Elevated suPAR levels are recognised as a marker of poor prognosis in HIV, tuberculosis, S.
pneumonia bacteraemia and malaria [389,474-476]. In our study, suPAR >9.25 ng/mL was
predictive of mortality in SAB with a specificity of 0.68 and sensitivity of 0.79. In HIV, suPAR >6 ng/ml is associated with high two-year mortality [477]. In tuberculosis, elevated suPAR levels associated with mortality, but the highest median levels reported were rather low 3.17 ng/mL [475]. In malaria, the highest median levels were slightly higher (7.9 ng/mL). In
S. pneumonia bacteraemia, even higher cut-off values (10-10.3 ng/mL) were prognostic
[389,472]. In critically ill patients, in (ICU) sepsis and in ventilator associated pneumonia predictive cut-off value for 28-day mortality was >12.9 ng/mL [478]. In another ICU study, suPAR level >12 ng/mL independently predicted mortality and suPAR levels were shown to remain stable for 10 days within survivors and non-survivors [479].
Although suPAR seems to predict mortality quite reliably in many kinds of infectious diseases, individual levels should be interpreted with caution. Even in our relatively small patient group, overlapping between groups of survivors and non-survivors was seen. This could be partly explained by the use of serum instead of plasma, since suPAR levels in serum are higher than in plasma, although suPAR concentrations in serum and plasma correlate well [480]. In addition, a number of ELISA methods to measure suPAR levels are available. Use of different ELISA methods in studies may partly explain differences in suPAR levels. One commonly used ELISA assay has been suPARnostic kit. This kit is validated to measure suPAR levels between 0.6 and 22 ng/mL. Whereas, another ELISA method can detect suPAR levels down to 15 pg/ml of suPAR [481]. In previous reports, suPAR levels in women are higher than men [52]. However, in our small study we could not detect any differences according to gender. Levels of suPAR increase with age [482] and we observed this in our analysis. In patients >65 years high suPAR levels (>9.25 ng/mL) were clearly more common compared to patients <65 years (Study IV, Table 3). Limitation of the Study IV was the small number of patients. In multivariate analysis number of covariates was limited due to insufficient number of patients. In addition, due to strong linkage, we could not determine the relationship between suPAR with age and fatal underlying diseases in the same multivariate analysis.
suPAR could possibly be used in predicting risk for poor outcome in SAB. Certain high suPAR level could help to allocate more resources to SAB patient at the greatest risk of dying. Such cut-off value due in SAB could be 9.25 ng/ml. However, low suPAR levels should not
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be interpreted as an impetus for a reduction in therapeutic intensity. CRP could help in clinical-decision making in SAB. CRP levels greater than 100 mg/L on days 1-4 four should lead to meticulous search, drainage and surgical treatment of deep infections and assessment of antibiotic therapy. CRP greater than 20 mg/L after two weeks of treatment should help raise clinical suspicion of deep infection at the latest.
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7. SUMMARY AND CONCLUSIONS
The effect of S. aureus on plg activation and prognostic factors in S. aureus bacteraemia can be summarised as follows:
I
An enhancing effect of both S. aureus bacterial cells and surface proteins on plg activation by SAK was discovered. In an analysis of S. aureus cell-surface proteins, threeplg-binding proteins were revealed as inosine 5´-monophosphate dehydrogenase, alpha-enolase and ribonucleotide reductase. In conclusion, we demonstrated that S. aureus enhanced plg activation by SAK. Furthermore, we could identify three previously unidentified tentative plg receptors of S. aureusII
In 430 SAB patients, predictive values of CRP in identifying fatal outcome and deep infections were determined. On day four, CRP >103 mg/L and on day 14, CRP >61 mg/L and WBC count >8.6 x109/L appeared prognostic of 30-daymortality in adjusted multivariate analysis. On the day of the positive blood culture, CRP >108 mg/L and on day 14 CRP >22 mg/L predicted the presence of deep infection. Compared to WBC count, CRP levels were superior in predicting mortality and presence of deep infection in SAB. These precise cut-off values of CRP might help diagnose patients with the complicated SAB.
III
In 145 S. aureus bacteraemia patients, the effect of CRP gene SNP on CRP levels in SAB was identified. The SNP rs3091244 A-minor allele was found to be a significant predictor of maximal CRP level in SAB. CRP gene SNPs were not associated with predisposing factors, underlying diseases, clinical outcomes, presence of deep infection or with the CRP levels at the time of the positive blood culture or one week after that. Together with a deep infection focus, carriage of the rs3091244 A- minor allele carriage was found to determine the maximal CRP during the acute phase in SAB.IV
The prognostic value of suPAR in 66 Staphylococcus aureus bacteraemia patients was identified. Day three suPAR levels >9.25 ng/mL predicted fatal outcome, and the difference in suPAR levels was observed up to 10 days after the onset of the disease. suPAR appeared not to be helpful in raising a suspicion of a deep infection.73
8. ACKNOWLEDGEMENTS
These studies were carried out at the Department of Bacteriology and Immunology, The Haartman Institute, University of Helsinki, during 1998-2002 and at the Department of Medicine, Division of Infectious Diseases, Helsinki University Hospital, during 2008-2017. I owe my warmest thanks to my supervisor Docent Asko Järvinen for his patience and guidance throughout this research project. Asko was never too busy for revising manuscripts and always encouraged me to continue this research project. My sincerest thanks go to my other supervisor Dr Eeva Ruotsalainen for her excellent, warm and enthusiastic guidance through my thesis project and for providing me the opportunity to continue studies with the S. aureus bacteraemia patient cohort. I am also extremely thankful to Docent Pentti Kuusela for introducing me to microbiological research as I started working on the first study of this thesis. Not only his vast expertise on virulence mechanisms of S. aureus but also sense of humor helped to cope.
This work started in late 1990s in Haartman Institute in Docent Pentti Kuuselas laboratory and I want to warmly thank all other members and collaborators of our study group and co-workers of that time. My sincerest thanks go to biomedical researchers and co-authors Jaana Tyynelä-Vesterinen, Jari Helin and Nisse Kalkkinen. For great technical assistance thanks goes to extremely skilful laboratory technician Sirpa Kuisma.
The third article, CRP gene work, was done in co-operation with the Department of Chronic Disease Prevention, Unit of Public Health Genomics, National Institute for Health and Welfare, Helsinki, Finland. I am especially grateful to co-authors of this study; Professor Markus Perola, Scientists Mervi Alanne and Annina Hautala for providing laboratory analyses and scientific advice on CRP gene SNP study. Professor Markus Perola provided invaluable help in statistical analyses and Scientist Annina Hautala did DNA sequencing and genotyping.
I want to thank co-author Christian Wandall Thorball for providing great expertise on suPAR analytics. Christian did suPAR laboratory analyses in the Clinical Research Centre, Copenhagen University Hospital, Hvidovre Hospital, Hvidovre, Denmark.
I want to express my gratitude to Docent Esa Rintala for his great advice and expertise in the clinical CRP article. Original idea for this article was largely based on discussions of our study group including Docent Esa Rintala. I also owe gratitude to statistician Jukka Ollgren. His expertise enabled reliable complex statistical analyses of the clinical CRP article. I want to thank Dr Erik Forsblom for invaluable peer support in statistics.
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I wish to warmly thank Professor Jaana Vuopio and Docent Pertti Arvola for their thorough and constructive review that greatly improved my thesis manuscript. I am also grateful to Derek Ho for great language revision.
My sincere thanks go to all colleagues at the Infectious Diseases Clinic of Helsinki for shared moments during years as a clinician and researcher. I want to warmly thank Docent Jussi Sutinen and Dr Eero Mattila for sharing their small work room with me, providing great tips on how to get an article submitted and above all for great and lasting friendship.
I wish to thank my parents. They have allways offered help, support and encouragement generously. Thank you also to my brother Timo for helping to loosen up sometimes. Finally, my deepest appreciation goes to my great family, my loving and understanding wife Hanna and our dear kids Oskari and Saara.
This work was financially supported by grants from the special Finnish governmental subsidy for health sciences (EVO-grant) and from Infektiolääkärit ry.
Helsinki, Tomi Mölkänen May 2017
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